Cells are required that amino acids are to be always incorporated thereinto as nutrition and such a function is carried by an amino acid transporter which is a membrane protein existing in cell membrane. The amino acid transporter is aligned in a specific site in each tissue in multicellular living things and plays an important role in expression of specific functions in each tissue.
A transport system asc is an amino acid transport system which transports small neutral amino acids mostly alanine, serine and cysteine and, originally, it was found in erythrocyte membrane and mentioned in many literatures. After that, its existence was confirmed in cultured cells as well (Christensen, Physiol. Rev., volume 70, page 43, 1990). The transport system asc is a transporter which is sodium-independent or, in other words, it does not need sodium ion for its function. Its transport substrate selectivity and transport characteristic have been known to have some differences depending upon cells and animal species.
The transport system asc shows a high affinity to a transport substrate such as alanine, serine or cysteine and, as a transport system similar thereto, there is a transport system C where small neutral amino acid such as alanine, serine or cysteine is a transport substrate as well but affinity to a transport substrate is low (Young et al., Biochem. J., volume 154, page 43, 1976; Young et al., Biochem. J., volume 162, page 33, 1977). The transport system C is believed to be a subsystem for the transport system asc. Sheep where the transport system C is genetically deficient was found, reduction in glutathione content in its erythrocytes was shown and the importance of incorporation of cysteine mediated by cell membrane in the production of glutathione was proved (Young, et al., Nature, volume 254, page 156, 1975).
However, in the conventional methods, it is difficult to analyze the details of transport of amino acid and analogs thereof mediated by the amino acid transport system asc and the functional role in vivo and there has been a demand to isolate gene of neutral amino acid transporter carrying a function of the amino acid transport system asc so as to make the detailed function analysis possible.
As to a small neutral amino acid transporter, there were cloned ASCT1 and ASCT2 (Kanai, Curr. Opin. Cell Biol., volume 9, page 565, 1997). However, they are sodium-dependent transporters and are entirely different from a sodium-independent amino acid transport system asc. In the meanwhile, glycine transporter and proline transporter were cloned (Amara and Kuhar, Annu. Rev. Neurosci., volume 16, page 73, 1993) but both of them transport only glycine and proline in a sodium-dependent manner and are different from the transport system asc.
cDNA of rBAT and 4F2hc which are type II membrane glycoproteins having only one transmembrane structure which is considered to be an activating factor of an amino acid transporter were cloned although they are not transporters per se and it was known that, when they were expressed in oocytes of xenopus, incorporation of basic amino acid together with neutral amino acid was activated (Palacin, J. Exp. Biol., volume 196, page 123, 1994).
With regard to a transporter which selectively transports neutral amino acid, there were cloned neutral amino acid transporter LAT1 (Kanai et al., J. Biol. Chem., volume 273, pages 23629–23632, 1998) and LAT2 (Segawa et al., J. Biol. Chem., volume 274, pages 19745–19751, 1999) corresponding to a transport system L. It was further shown that LAT1 and LAT2 functioned only when they coexist together with a cofactor 4F2hc. Both are not dependent on Na+ but LAT1 shows an exchange transport activity for transporting the large neutral amino acids such as leucine, isoleucine, valine, phenylalanine, tyrosine, tryptophane, methionine and histidine and LAT2 has a wide substrate selectivity transporting the small neutral amino acids such as glycine, alanine, serine, cysteine and threonine in addition to large neutral amino acids. However, even they are different from the amino acid transport system asc in terms of substrate selectivity.
With regard to proteins analogous to the neutral amino acid transporters LAT1 and LAT2, there were cloned the above-mentioned y+LAT1 and y+LAT2 having a function of a transport system y+L transporting the neutral amino acids and basic amino acids (Torrents et al., J. Biol. Chem., volume 273, pages 32437–32445, 1998). It was also shown that both y+LAT1 and y+LAT2 functioned only when coexisted together with the cofactor 4F2hc. y+LAT1 and y+LAT2 mainly transport glutamine, leucine and isoleucine as neutral amino acids and are different from the amino acid transport system asc in terms of the substrate selectivity.
With regard to a transporter demanding the cofactor 4F2hc for the expression of the function, there was cloned xCT which is a protein analogous to the neutral amino acid transporters LAT1 and LAT2 (Sato et al., J. Biol. Chem., 274: 11455–11458, 1999). The xCT transports cystine and glutamic acid and is different from the amino acid transport system asc in terms of the substrate selectivity.
With regard to a transporter demanding other cofactor rBAT having a structure analogous to 4F2hc for expressing the function, there was cloned BAT1 which is a protein analogous to the neutral amino acid transporters LAT1 and LAT2 (Chairoungdua et al., J. Biol. Chem., 274: 28845–28848, 1999). BAT1 transports cystine, neutral amino acids and basic amino acids and is different from the amino acid transport system asc in terms of substrate selectivity.
As such, molecular substances of the transporters which function upon linking to 4F2hc and rBAT were made clear and it was clarified that there was a group of transporters which achieve a transporting function when a molecular complex is formed with the type II glycoprotein.